TAK1 (encoded by Map3k7) is a mitogen-activated protein kinase kinase kinase (MAP3K), which activates the transcription factors AP-1 and NF-kappa B in response to receptor activator of NF-kappa B ligand (RANKL) stimulation, thus constituting a key regulator of osteoclast differentiation. Here we report the functional relevance of the kinase activity of TAK1 in the late stage of osteoclast differentiation in vivo using Ctsk-Cre mice and TAK1 mutant mice in which the TAK1 kinase domain was flanked by loxP. The Map3k7(flox/kd)Ctsk(Cre/+) mice displayed a severe osteopetrotic phenotype due to a marked decrease in osteoclast number. RANKL-induced activation of MAPK and NF-kappa B was impaired in the late stage of osteoclast differentiation. The absence of suppressive effect of an administered NF-kappa B inhibitor on the late stage of osteoclastogenesis led us to investigate unknown TAK1 targets in osteoclast differentiation. We performed a phosphoproteomic analysis of RANKL-stimulated osteoclast precursor cells from Map3k7(flox/kd)Ctsk(Cre/+) mice, revealing multiple targets regulated by TAK1 during osteoclastogenesis. Thus, TAK1 functions as a critical regulator of the phosophorylation status of various cellular proteins that govern osteoclastogenesis. (C) 2015 Elsevier Inc. All rights reserved.